Basophil degranulation test

ABSTRACT

The present invention concerns a method for determining the degranulation activity of basophilic granulocytes in a sample as well as a reagent kit that is suitable for this. Furthermore the invention also concerns a method for diagnosing allergic hypersensitivity and for monitoring the response to a hyposensitization therapy.

FIELD OF THE INVENTION

The present invention concerns a method for determining thedegranulation activity of basophilic granulocytes in a sample as well asa reagent kit that is suitable for this. In addition the invention alsoconcerns a method for diagnosing allergic hypersensitivity and theresponse to a hyposensitization.

BACKGROUND AND PRIOR ART

The classical classification of allergies according to Coombs and Gellstill largely applies nowadays as a basis for understanding the allergictype 1 reaction of the immediate type and is clearly defined by anIgE-mediated immune response. T cells with a particular cytokine pattern(interleukin 4 and interleukin 5) play a special role in this. As aresult B lymphocytes are stimulated to switch to IgE production whichonly occurs at very low levels in the serum and only develops its fataleffect after cell binding to high affinity receptors and subsequent celldegranulation. The cytokines, in particular interleukin 5, additionallycause an increased maturation of promyelocytes into eosinophilicgranulocytes which cause delayed damage especially to the bronchialsystem by the release of toxic proteins from their granula. Thephysiological relevance of the type 1 allergy is still completelyunclear as well as the reason why the T cells secrete a cytokine patternfor an IgE response.

The determination of specific IgE antibodies in the serum (RAST, CAPetc.) is well established and is especially helpful when known allergensneed to be tested or when a skin test is dangerous or technicallyimpossible for example in skin diseases or under the influence of drugs.In these methods the allergens are bound covalently or with highaffinity to matrices with a large surface (sponges, beads, paper discs),subsequently incubated with patient serum and then, after washing, adetection is carried out using a radioactive or enzyme-labelledanti-human-IgE antibody. The amount of bound IgE is statedsemiquantitatively in so-called RAST and CAP classes or quantitativelyin kU IgE/l. The evaluation can be carried out radiometrically (RAST) orfor example, fluorimetrically (CAP, enzyme with a fluorogenicsubstrate).

The advantages of these tests are that they are easy to carry out andthere is a good correlation with the gold standard skin test foraeroallergens and so-called atopens. However, serious disadvantages mustbe accepted. These disadvantages are for example the lack of rareallergens and often epitope changes due to coupling of the allergens tothe matrix. In addition a carrier molecule such as human serum albuminis required for smaller allergens (haptens). Further difficulties arecaused by the fact that the specificities of serum IgE andmembrane-bound IgE on basophils and mast cells are not necessarily thesame since only a slow exchange takes place. Also the threshold ofdegranulation readiness (releasability) is not detected. Correlationwith the response to a hyposensitization in the form of an IgE decreaseis only fairly satisfactory for insect venom allergens i.e. in themajority of cases there is no explanation. Furthermore the specific IgEvalues must be put into perspective in the case of atopic patients witha high IgE level i.e. the total IgE level of the patient must be known.The severity of the clinical picture hardly correlates with the level ofthe specific IgE concentration.

Ready-made test strips have been developed as a simplified version ofthis method on which the various allergens are applied. They areevaluated by comparing the blue colour that forms with a colour scale.Inhalation antigens which produce a reaction in the higher RAST classesare readily detected. Otherwise there is little comparative data. Thetest is primarily conceived for allergists in private practice who donot have a large laboratory.

Due to the problems mentioned above in determining specific serum IgE,tests have been developed in which the basophilic granulocytes aredegranulated after stimulation with an allergen and subsequently thereleased components of the granula are determined in the supernatant.Such components are for example histamine (Immunotech Co. IBL),leukotrienes (CAST-ELISA, Biermann Co.) or tryptase (Pharmacia Co.).These assays are designed as enzyme immunoassays in a batch procedureand require a duration of two days due to overnight incubation steps. Afurther disadvantage with regard to the histamine determination is theacylation step for the released histamine as well as a frequentlyexcessively high histamine content of the allergen solutions due tobacterial contaminations. Furthermore for economic reasons it isnecessary to collect the supernatants of several patients which delaysthe assessment. The degranulation must, however, be carried out on freshwhole blood on the day of blood withdrawal.

Therefore attempts have already been made to detect the degranulationcytometrically. In one approach the absorption of basophilic granula wasdetermined with a Technicon H6000 measuring instrument. In this case themeasurement points were counted by hand on the screen (Nilsson, Eur. J.Haematol, 45 suppl 53, 50-54, 1990). Alternatively the axial loss oflight is measured after staining with toluidine blue in an Orthocytofluorograph (Nakagawa et al. (Allergy 36, 39-47, 1981). A furtherapproach is to carry out a flow-cytometric two colour immunofluorescencemethod in which differently labelled anti-CD45 and anti-IgE antibodiesare used (Gane et al., Cytometry 19, 361-365, 1995). This method isbased on a relative increase of the expression density of CD45 and areduction of the IgE antibody expression in degranulation. However, adisadvantage of this method is that the change in the CD45 expressiondensity is only small.

None of these cytometric methods has been introduced for routine use dueto the said disadvantages since they are tied to special, not generallyavailable instruments or the signal differences are very slight.Furthermore it is difficult to measure the basophils since they are rarein the blood and are very difficult to distinguish from other leucocyteson the basis of scattered light properties alone.

SUMMARY OF THE INVENTION

Hence the object that forms the basis of the present invention is toprovide a new method for determining the degranulation activity ofbasophilic granulocytes which can be carried out simply and rapidly andcan be performed with conventional flow cytometers which are widely usedin hospitals and laboratory practices.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the evaluation of a test control according to the inventionfor a negative control

FIG. 2 shows the evaluation of a test according to the invention for apositive control

FIGS. 3-5, inclusive, show comparison of tests according to theinvention, with a histamine release test of prior art.

FIG. 6 shows that evaluation of a test according to the invention withprestiinulation with IL-3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This object is achieved by a method for determining the degranulationactivity of basophilic granulocytes in a sample, wherein

a) the sample is mixed and incubated with a test substance,

b) the sample is mixed and incubated with a first binding molecule whichcan bind to cell-bound IgE antibodies or high affinity receptorsthereof, and with a second binding molecule which can bind to a surfacemolecule which appears de novo on the surface of basophilic granulocytesduring degranulation, in which the first binding molecule and the secondbinding molecule carry different marker groups that can be detectedconcurrently or can each bind to different marker groups that can bedetected concurrently, and

c) the labels of the binding molecules are determined separately and thedegranulation activity in the sample is determined qualitatively or/andquantitatively on the basis of this determination.

In the method according to the invention the basophils are firstlyunequivocally determined via the marker using a first specific bindingmolecule e.g. an antibody. This first binding molecule binds tocell-bound IgE antibodies or high affinity receptors thereof. Onlybasophils have a high surface density of IgE molecules which are boundto high affinity receptors (Fc_(ε)R1). IgE-specific or receptor-specificantibodies are preferably used which do not lead to a cross-linking ofthe IgE.

A further special feature of the method according to the invention isthat a second binding molecule is used which can bind to an antigen thatonly appears on the surface as a result of degranulation (de novoexpression). An example of such an antigen is the preformed lysosomalprotein gp55 which does not appear on the surface until the granulamembrane has fused with the cytoplasmic membrane during degranulationand can be detected there.

The term “test substance” in the present invention is understood as anymolecule which can cause an activation of basophilic granulocytes ordegranulation. The test substance is usually an allergen such as plantpollens, an insect venom such as bee or wasp venom, a drug such aspenicillin or a latex allergen. In addition “test substance” also refersto other biological and synthetic substances which can cause such anactivation and can be used in the sense of a positive control. Anexample of this is the chemotactic peptide fMLP.

In one embodiment of the method according to the invention the sample ispreincubated with a stimulant such as a cytokine which amplifies thedegranulation activity. The amount of cytokine is preferably 0.1-100 ngper test mixture. This preincubation leads to a sensitization of thebasophilic granulocytes which reduces the threshold for degranulationreadiness (releasability). The stimulant used is preferably a lymphokinesuch as an interleukin or a growth factor such as a colony stimulatingfactor (CSF). IL-3 or GM-CSF are preferred. Preincubation with thecytokine is usually carried out before step (a) of the method accordingto the invention i.e. before adding the allergen, but it can also becarried out simultaneously with the allergen incubation.

Incubation with an allergen as the test substance in the case of asample derived from an allergy patient leads to an activation of thebasophilic granulocytes and thus to a degranulation the extent of whichcorrelates with the extent of the allergic reaction. The first bindingmolecule serves to define the basophilic granulocytes and the secondbinding molecule serves to define that portion of the basophilicgranulocytes in which a degranulation has taken place. The relativeproportion of the degranulated granulocytes can thus be determined in asimple manner from the ratio between the cells that are positive for thefirst and second binding molecule and the cells which are only positivefor the first binding molecule.

The antigen recognized by the second binding molecule is usually aprotein or a glycoprotein which only appears on the surface as a resultof degranulation and thus allows an unequivocal differentiation betweendegranulated and non-degranulated granulocytes. The surface molecule ispreferably gp55.

Any biological substances can be used as the first and second bindingmolecule provided they bind specifically either to IgE antibodies or totheir high affinity receptors or to a degranulation-specific surfacemolecule. An antibody or an antibody fragment is preferably used as afirst or/and a second binding molecule. Monoclonal antibodies arepreferred but polyclonal antibodies are also suitable. In particular forthe first binding molecule, those antibodies or antibody fragments aresuitable which have a high specificity but low cross-linking ordegranulation properties (e.g. Fab fragments). Antibodies or antibodyfragments against the Fc_(ε) receptor are particularly suitable as thefirst binding molecule since the cells in only a small number of thepatients are inadequately recognized and activated by anti-IgE.

Furthermore it is preferred in the method according to the invention toterminate the degranulation after a defined incubation period with theallergen and the first and second binding molecule. This can for examplebe achieved by fixing the leukocytes. Furthermore it is preferable tolyse the erythrocytes after the incubation. Lysis of the erythrocytesand fixing of the leukocytes can also optionally be carried out in onestep. Hence a solution is added to the sample which for example containsa high concentration of ammonium chloride and EDTA. Solutions whichcontain formaldehyde and/or ethylene glycol are also suitable. Apreferred solution for carrying out lysis and fixation is a commerciallyavailable solution under the trade name FACS lysing solution (BectonDickinson). A wash step is optionally carried out after the fixation.

In addition to the determination of IgE and de novo expressed surfacemolecules, the method according to the invention can also comprise thedetermination of one or several additional parameters. Thus the samplecan be additionally incubated with a third binding molecule which iscapable of binding to the surface molecule CD45 and carries a markergroup or is capable of binding to a marker group which can be determinedseparately in addition to the marker groups of the first and secondbinding molecule. The third binding molecule is preferably an anti-CD45antibody or a fragment of such an antibody.

The additional determination of CD45 whose expression is increased afteractivation of the basophilic granulocytes further increases the accuracyof the determination.

A further parameter that can also be determined in the method accordingto the invention is the light that is scattered sideways which increasesduring degranulation of basophils.

The binding molecules used in the method according to the invention cancarry a marker group or can bind to a marker group. If the bindingmolecule carries a marker group i.e. is directly labelled, it ispreferably present as a covalent conjugate with the marker group. On theother hand the binding molecule can also be indirectly labelled i.e. itdoes not directly carry a detectable marker group but can bind to afurther substance which carries the marker group. Examples of indirectlylabelled binding molecules are hapten-coupled antibodies which can bindto an anti-hapten antibody carrying a marker group. The coupling ofmarker groups and haptens to biological binding molecules, in particularto antibodies or antibody fragments, is known to a person skilled in thefield of immunological test procedures and does not therefore requirefurther elucidation. Directly labelled binding molecules are preferredfor the method according to the invention.

Essentially all known markers in the field of diagnostic detectionmethods are suitable as marker groups for the binding molecules, wherethe only prerequisite is that the individual markers can be determinedconcurrently. Fluorescent markers are preferred which each have adifferent spectral emission spectrum e.g. fluorescein, phycoerythrin(PE) and peridinium-chlorophyll-A protein (Per CP) or a PE-cyanine 5tandem conjugate (e.g. cychrome). The determination is preferablycarried out in a flow cytometer.

According to another preferred embodiment of the present method a DNAdye which can be determined in addition to the marker on the anti-IgEantibody and receptor is added and determined as a further parameter.The DNA dye serves to stain the cell nuclei and thus to excludethrombocytes and unlysed erythrocytes. A fluorescent dye such as7-aminoactinomycin D or propidium iodide is preferred.7-aminoactinomycin D is particularly preferred.

The sample used in the method according to the invention is a body fluidwhich contains granulocytes. A particular advantage is that the methodaccording to the invention can be used on whole blood without requiringa cell isolation of basophilic granulocytes. Heparinized whole blood ispreferred as the sample and it is generally derived from a human donoralthough other samples from mammals such as rodents can also be used.

The method according to the invention is very simple and rapid. It canbe carried out on any commercial flow cytometer which are present in allmedium and large hospitals and laboratories.

The method conditions can be varied depending on the test format used.When using fluorescent markers and a flow cytometric detection of themarkers, suitable conditions for step (a) i.e. incubation with the testsubstance have turned out to be a time period of 5 to 60 min, preferablyof 20 to 40 min at a temperature of 25 to 40° C., preferably of 35 to39° C. A 30 min incubation at about 37° C. gives good results. Step (b)is preferably carried out in the cold in order to avoid unspecificdegranulation and suitable conditions have turned out to be atemperature of 0 to 10° C., preferably a temperature of 0 to 5° C. for atime period of 10 to 40 minutes, preferably of 5 to 30 minutes. If afixation is carried out after step (b), a further cooling of the sampleis not essential since the fixation prevents further degranulation. Step(c), i.e. the determination of the markers, is carried out following theincubations and optionally lysis and subsequent wash steps. In thisconnection the measurement sample is adequately stable for a period ofat least one hour, preferably about two hours or more.

As is apparent to a person skilled in the art, it is expedient tomeasure suitable controls in a determination by the inventive method inwhich a mixture without the test substance is suitable as the negativecontrol and a substance that activates the granulocytes such as fMLP issuitable as a positive control. Furthermore it is advantageous to alsomeasure a sample whose values are essentially in the normal range suchas a body fluid from a healthy normal test person.

A further subject matter of the present invention is a reagent kit forthe determination of the degranulation activity of basophilicgranulocytes in a sample. The reagent kit contains a first bindingmolecule which can bind to cell-bound IgE antibodies or their highaffinity receptors and a second binding molecule which can bind to asurface molecule which appears de novo on the surface of basophilicgranulocytes during degranulation in which the first and the secondbinding molecule carry different marker groups that can be detectedconcurrently or each can bind to different marker groups that can bedetected concurrently. Optionally the reagent kit can contain a thirdbinding molecule as an additional detection reagent which can forexample bind to the CD45 surface molecule and carries a marker groupwhich can be detected in addition to the markers of the first and secondbinding molecule or can bind to such a marker group. The reagent kit canalso contain a DNA dye. Further optional components of the reagent kitare test substances i.e. various allergens or positive controlsubstances such as fMLP. In addition the reagent kit can also containstimulants such as cytokines or/and reagents for lysing erythrocytesor/and for fixing leukocytes.

Furthermore the kit can additionally contain a reagent for lysingerythrocytes and/or for fixing leukocytes as well as optionallyconventional buffers, auxiliary substances or additives.

A preferred reagent kit contains for example at least one testsubstance, an anti-gp55-FITC antibody, an anti IgE-PE antibody, a FACSlysing solution as well as optionally 7-aminoactinomycin D or/and ananti-CD45-PerCP antibody and a stimulant such as IL-3 or GM-CSF.

Yet a further subject matter of the invention is a method for diagnosingthe allergic hypersensitivity of a patient or the response to ahyposensitization which is characterized in that the degranulationactivity of basophilic granulocytes is determined in a sample from apatient by means of the method according to the invention using one orseveral allergens and a positive control as test substances, thedegranulation activity in a negative control sample is determined in anadditional test mixture and the presence or the absence of an allergichypersensitivity or the success or failure of a hyposensitizationtherapy is determined by comparing the values that are obtained.

An appropriate negative control sample is a sample from the same patientto which no test substance has been added in step (a) of the methodaccording to the invention and preferably a stimulated sample of ahealthy normal test person is also assayed as a second negative controlsample.

An additional subject matter of the invention is the use of anti-gp55antibodies in a method for determining the degranulation activity ofbasophilic granulocytes and the use of the method and reagent kitaccording to the invention for the diagnosis of allergichypersensitivity and for monitoring the response to a hyposensitizationtherapy.

EXAMPLE 1

Peripheral blood (heparinised) of the patient is incubated with variousconcentrations of the suspected allergen at 37° C. In the case of acorresponding sensitization this leads to a degranulation of thebasophilic granulocytes and to an activation which is primarilycharacterized by expression of the gp-55 antigen. In addition theexpression density of the IgE receptor decreases and the density of CD45on basophils increases. The side scattered light increases (loss ofabsorbing granula) and the number of basophils in the scattered lightgate for lymphocytes decreases. The detection is carried out by a threecolour immunofluorescence and flow cytometric analysis. The chemotacticpeptide fMLP serves as a positive control and a buffer solution (PBS) isused as the negative control. Antihistamine administration inhibits thetest.

1. Material

1.1 Reagents

Sheath liquid: FACS-flow (Becton Dickinson Co. order No. 342003) oranother carrier liquid with a low intrinsic fluorescence.

FACS lysing solution, 10× concentrate (Becton Dickinson Co. order No.349202).

Wash solution: PBS (phosphate buffered saline without calcium andmagnesium) as a ready-to-use solution or as salts (e.g. Biochrom,Berlin) or PHAGO-BURST wash solution (ORPEGEN Pharma Co., Heidelberg).

Chemotactic Peptide fMLP (2000×) (ORPEGEN Co.) diluted in PBS.

For the calibration of quantitative measurements: Calibrite calibrationparticles, Becton Dickinson, order No. 349502.

1.2 Monoclonal Antibodies

a) anti gp55-FITC, (ORPEGEN Pharma), order No. 1464 (prediluted 1:4 inthe ready-made combination)

b) anti IgE-PE (ORPEGEN Pharma), order No. 1465 (prediluted 1:40 in theready-made combination)

c) anti CD45 PerCP (Becton Dickinson) or CD45 cychrome (Pharmingen),order No.

1.3 Allergens

ALK depot SQ (Scherax) mixture of 6 grasses+rye 100,000 U/ml, order No.145a/90.90c

latex allergen (ALYOSTAL ST-IR) order No. 0903

ALK-prick SQ (Scherax) bee venom, order No. 222a/85a, 300 μg/ml

ALK-prick SQ (Scherax) wasp venom, order No. 223a/85a, 300 μg/ml

Allergopen (penicillin G, PPL and derivative MDM), order no. 162a/81,

penicillin “Grünenthal” 1 mega, PZN-7803133 mite allergen from the GeerLabs company among others.

2. Procedure

2.1 Sample Preparation

The sample to be examined is divided into aliquots of 100 μl in eachcase. In the case of high leukocyte numbers (WBC of 10-20,000 C/μl) itis possible to use a smaller sample volume e.g. 50 μl. Multiple testmixtures are not necessary.

2.2 Preparation of the Allergen Dilutions

ALK-depot SQ (Scherax) mixture of 6 grasses+rye is used atconcentrations of 10,000, 1,000, 100 and 10 U/ml. ALK-prick SQ (Scherax)bee venom and ALK-prick SQ (Scherax) wasp venom are used atconcentrations of 10, 1, 0.1 and 0.01 μg/ml. Latex allergens, PPL(allergopen) and MDM (allergopen) are used in dilutions of 1:10, 1:100and 1:1,000. Penicillin Grünenthal is used at concentrations of 100, 10,1 and 0.1 μg/ml. PBS serves as a negative control and fMLP at aconcentration of 10⁻⁷ M (i.e. 1:2,000 PBS) is used as the positivecontrol. A sample from a healthy normal test person is also assayed as afurther control.

2.3 Incubation with Allergen

100 μl of each of the allergen dilutions is added to an aliquot of thesample, mixed and incubated for 30 minutes at 37° C. in a water bath.Then the mixtures are placed on ice.

2.4 Addition of Antibodies

The following antibody solutions are then added.

a) 20 μl anti gp55-FITC (prediluted)

b) 20 μl anti IgE-PE (prediluted)

c) 10 μl anti CD45-cychrome or 20 μl CD45-PerCP

After antibody addition it is well mixed (vortex) and incubated for 20minutes on ice while protected from light.

2.5 Lysis and Fixing

2 ml FACS lysing solution (erythrocyte lysis and leukocyte fixation) isthen added, it is mixed (vortex) and incubated for 10 minutes at roomtemperature while protected from light. Subsequently it is centrifugedfor 5 min at 1300 rpm or 300 g and decanted.

2.6 Washing the Cells

The cells are washed by adding 3 ml wash solution (PBS), centrifuging (5min at 1300 rpm or 300 g) and decanting.

2.7 Measurement

The sample is measured by flow cytometry for example on an FACS scaninstrument. The stability of the measured sample is presumably ≧2 hourson flaked ice in the dark.

The instrument settings (“BASO-SET” in the program CellQuest®) can beselected as follows.

Expected Parameter Amplifier value resolution FSC lin E00 (2.0) 256channels SSC lin 360 256 channels FL1 log, 4 decades 620 256 channelsFL2 log, 4 decades 500 256 channels FL3 log, 4 decades 620 256 channelstrigger on 100 FSC compensation FL1-% FL2  0.9 FL2-% FL1  21 FL2-% FL3 0.2 FL3-% FL2  29

filter combinations (not variable in the FACScan):

Parameter spectrum fluorochrome filter FL1 fluorescence 1 green FITC, TOBP530 +/− 15 nm fluorescence FL2 fluorescence 2 orange PE, PI BP585 +/−22 nm fluorescence FL3 fluorescence 3 red PerCP, red613, LP > 650 nmfluorescence Cy5, PI

In the SSC/IgE dot plot, a region is set around the cells with low andhigh side scattered light and strong IgE expression. These cells areoptionally displayed in the dot plot FL3 (CD45) versus FL2 (IgE) as partof the validation and the x and y mean values of this population areevaluated and the FL1 histogram (gp 55). The markers are set such thatthe evaluation region covers the region of the gp55 positive basophils(the PBS test mixture serves as a negative control). The mean value FL1is observed in all (also gp55-negative) cell populations. If possible,at least 1000 cells of the subclass of interest (in this case basophils)are recorded. In the positive test mixture a bimodal distribution isformed in which the activated basophilic granulocytes represent anindividual population. It is therefore possible to view the percentagewhich is adequate for evaluating the test result.

3. Evaluation

The evaluation of a negative control containing PBS is shown in FIG. 1.The evaluation of a positive control containing fMLP is shown in FIG. 2.

The following Table 1 shows typical values where all parameters shouldbe taken into consideration as a plausibility check. A comparisonbetween the negative control (PBS) and positive control (fMLP) showsthat the percentage of basophils remains essentially constant, the CD45expression density increases after activation and the IgE densitydecreases slightly. The percentage of gp55-positive basophilicgranulocytes and the mean gp55 value increases strongly afteractivation.

assay % basos mean CD45 mean IgE % gp55 mean gp55 PBS 2.50 134 1032 3.3 33 fMLP 2.35 349  981 30.6 369

A comparison of the diagrams for CD45 and gp55 for the positive andnegative control shows that a considerably higher allergen-specificallyinduced increase of expression occurs in the case of gp55.

Results of the method according to the invention are shown in FIGS. 3-6.FIG. 3 shows a comparison of the method according to the invention (gp55or CD63 expression) with a conventional histamine release test for thedetermination of a grass pollen allergy. Corresponding data are shown inFIG. 4 for the determination of a bee venom allergy and in FIG. 5 forthe determination of a wasp venom allergy. An example of a latexallergen determination shown in FIG. 6 shows that the degranulationactivity of basophils can be considerably increased by prestimulationwith a cytokine like IL-3.

Legends

FSC=forward light scatter=narrow angle forward light scatter=cell size

SSC=side scatter=orthogonal light scatter=cell granularity

algE-PE=IgE-PE=monoclonal antibody against immunoglobulin E, directlyconjugated with the fluorescent dye phycoerythrin.

gp55-FITC=monoclonal antibody against the lysosomal glycoprotein with amolecular weight of 55 kD, directly conjugated with the fluorescent dyefluorescein.

CD45 cychrome=monoclonal antibody against the common cell surfaceglycoprotein of leukocytes directly conjugated with the tandem dyePE-cyanin 5.

R1, R2, R3=regions in which certain cells are located and which functionas a logical gate. In this case they are the typical regions forbasophilic granulocytes.

M1=marker region in which the cells which react positively to theantigen gp55 are located (degranulated basophils).

What is claimed is:
 1. A method for the determination of degranulationactivity of basophilic granulocytes in a whole blood sample, comprising:a) preincubating the whole blood sample with a stimulant that amplifiesdegranulation activity, b) incubating said whole blood sample with atest substance, c) incubating said whole blood sample witha firstbinding molecule which binds to either cell-bound IgE antibodies or tohigh affinity receptors thereof, and with a second binding moleculewhich binds to a CD63 molecule, wherein said first binding molecule andsaid second binding molecule (i) carry different marker groups which aredetectable concurrently, or (ii) bind to different marker groups whichare detectable concurrently, or (iii) one of said binding moleculescarries a marker group and the other binds to a different marker group,said marker groups being detectable concurrently; d) lysing erythrocytesin said whole blood sample, and e) separately determining the markergroups of said first and second binding molecules, wherein degranulationactivity in said whole blood sample is determined from the ratio betweenthe cells that are positive for both the first and second bindingmolecule and the cells which are only positive for the first bindingmolecule.
 2. The method of claim 1, wherein said stimulant is acytokine.
 3. The method of claim 2,wherein said cytokine is IL-3 orGM-CSF.
 4. The method of claim 1, wherein at least one of said bindingmolecules is an antibody or an antibody fragment.
 5. The method of claim1, wherein said first binding molecule is an anti-IgE antibody, afragment thereof, an anti-Fce_(ε)RI antibody or a fragment thereof. 6.The method of claim 1, wherein said second binding molecule is ananti-CD63 antibody or a fragment thereof.
 7. The method of claim 1,further comprising fixing leukocytes in said whole blood sample prior toseparately determining said marker groups.
 8. The method of claim 7,further comprising washing said whole blood sample after fixingleukocytes.
 9. The method of claim 1, further comprising incubating thewhole blood sample with a third binding molecule which binds to CD45molecules, wherein said third binding molecule carries a marker group orbinds to a marker group that is detectable separately from the markergroup of the first and second binding molecules.
 10. The method of claim1, wherein said binding molecules are directly labeled.
 11. The methodof claim 1, wherein said binding molecules carry fluorescent markers.12. The method of claim 11, comprising flow cytometry.
 13. The method ofclaim 1, further comprising incubating said whole blood sample with adetectable DNA dye.
 14. The method of claim 13, wherein said detectableDNA dye is a fluorescent dye.
 15. The method of claim 14, wherein thefluorescent dye is 7-amino actinomycin D.
 16. The method of claim 1,wherein said whole blood sample has been heparinized.
 17. The method ofclaim 1, wherein said whole blood sample is a sample taken from a human.18. The method of claim 1, comprising incubating said whole blood sampleand test substance for 5-60 minutes at a temperature of 25-40° C. 19.The method of claim 18, comprising incubating for 20-40 minutes at atemperature of35-39° C.
 20. The method of claim 1, comprising incubatingsaid whole blood sample and said binding molecules for 10-40 minutes ata temperature of 0-10 ° C.
 21. The method of claim 1, wherein secondbinding molecule is an anti-CD63 antibody, labeled with FITC.
 22. Themethod of claim 1, wherein said sample is taken from a patientundergoing hyposensitization therapy.
 23. Reagent kit useful indetermination of degranulation activity of basophilic granulocytes, inwhole blood, comprising (i) a first component which is a first bindingmolecule which binds to cell bound IgE antibodies or high affinityreceptors thereof, (ii) a second component which is a second bindingmolecule which binds to a CD63 molecule, wherein said first and secondbinding molecules either carry different marker groups that aredetectable concurrently or bind to different marker groups that aredetectable concurrently, (iii) a third component which is a stimulantthat amplifies degranulation activity of basophilic granulocytes, and(iv) a fourth component which is a reagent for lysing erythrocytes. 24.The reagent kit of claim 23, further comprising a third binding moleculewhich binds to a CD45 surface molecule and either carries a marker groupwhich is detectable in addition to said markers of said first and secondbinding molecules or binds to said marker group.
 25. The reagent kit ofclaim 23, further comprising a DNA dye.
 26. The reagent kit of claim 23,further comprising a reagent for fixing leukocytes.
 27. The reagent kitof claim 23, wherein said marker groups are flourescent labels.
 28. Thereagent kit of claim 23, wherein component (ii) is an anti-CD63-FITCantibody, or component (i) is an anti-IgE-PE antibody, or component (iv)is a FACS lysing solution, or the kit further comprises a testsubstance.
 29. The reagent of kit of claim 23, further comprising atleast one of 7-aminoactinomycin D, or an anti-CD45-PerCP antibody. 30.The reagent kit of claim 23, further comprising a buffer, an auxiliarysubstance or an additive.
 31. A method for diagnosing allergichypersensitivity in a patient comprising: (a) contacting a whole bloodsample from said patient with (i) at least one allergen, (ii) a firstbinding molecule which binds to either cell-bound IgE antibodies or highaffinity receptors thereof, and (iii) a second binding molecule whichbinds to a CD63 molecule, wherein said first and second bindingmolecules either carry different marker groups that are detectableconcurrently or bind to different marker groups that are detectableconcurrently, (b) determining said marker groups separately, and (c)comparing the values obtained with said allergen to the values obtainedwith a positive control and a negative control, wherein allergichypersensitivity in said patient is indicated when the value obtainedwith said allergen is higher than the value obtained with the positivecontrol.
 32. A method for determining degranulation of basophilicgranulocytes in a whole blood sample comprising contacting said samplewith an antibody that binds to CD63 molecules, and determining bindingof said antibody, wherein the level of binding is a measurement ofdegranulation of basic granulocytes in said sample.
 33. A method fordetermining a patient's response to hyposensitization therapycomprising: (a) contacting a whole blood sample from said patient with(i) at least one allergen, (ii) a first binding molecule which binds toeither cell-bound IgE antibodies or high affinity receptors thereof, and(iii) a second binding molecule which binds to a CD63 molecule, whereinsaid first and second binding molecules either carry different markergroups that are detectable concurrently or bind to different markergroups that are detectable concurrently, (b) determining said markergroups separately, and (c) comparing the values obtained with saidallergen to the values obtained with a positive control and a negativecontrol, wherein said patient has responded to hyposensitization therapyif the value obtained with said allergen is lower than the valueobtained with the positive control.